Nano-enzyme-sensitized electrochemiluminescence immune biosensor as well as construction method and application of nano-enzyme-sensitized electrochemiluminescence immune biosensor

Abstract

The invention discloses a nano-enzyme-sensitized electrochemical luminescence immune biosensor as well as a construction method and application thereof. The biosensor comprises a three-electrode system of an immune electrode, a reference electrode and a counter electrode, and a solution containing luminol and hydrogen peroxide is taken as an electrolyte solution; the immunoelectrode is obtained by covalently immobilizing a tumor marker antibody and closing a non-specific site on an electrode confined to Co3O4 nano-enzyme. On the basis of an ECL signal of an SNF confinement Co3O4 nano-enzyme sensitization luminol-H2O2 system, an antibody on an immune recognition interface in detection can specifically capture a steric hindrance effect caused by a tumor marker and an immune complex, so that the ECL signal is reduced, and a detection effect is achieved. The Co3O4 nano-enzyme is generated in situ in a nano-channel, the activity of pseudo-peroxidase is enhanced while the Co3O4 nano-enzyme is stabilized, and the Co3O4 nano-enzyme is used as a co-reaction accelerant to sensitize ECL signals. The sensor disclosed by the invention is used for detecting tumor markers and has the advantages of high sensitivity and low detection limit.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a nanozyme-sensitized electrochemiluminescent immunoassay biosensor and a construction method and application thereof. Background Art

[0002] Sensitive detection of tumor markers is of great significance for early diagnosis and subsequent monitoring and treatment of cancer. Alpha-fetoprotein (AFP) is one of the main specific tumor markers associated with liver cancer. The AFP content in normal human serum is usually lower than 25 ng / mL, while the serum AFP content in liver cancer patients is significantly increased. Therefore, detecting the AFP concentration in serum is of great significance for the diagnosis of primary liver cancer. At present, there are several methods for detecting alpha-fetoprotein in serum: such as electrochemical method, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay and chemiluminescence. Among them, the immune sandwich detection mode often requires antibodies labeled with enzymes, which will cause the modified biological molecules to denature, and it is also costly and time-consuming. Compared with sandwich immunoassay, antibody-free labeling immunosensor does not require a second antibody, and the construction method is more convenient.

[0003] Electrochemiluminescence (ECL) is a detection technology that combines electrochemistry and chemiluminescence. ECL has the advantages of low background noise, simple operation, and wide linear range, and has received increasing attention in the fields of imaging, biosensing, and chemical detection. Among them, the ECL system based on the luminol luminophore-co-reactant system has attracted widespread attention from scientific researchers due to its low excitation potential and wide range of analytical applications. However, the luminol system is still limited by the low ECL quantum yield. Therefore, it is crucial to improve the ECL intensity of the luminol system on conventional electrodes.

[0004] Nanoparticles (NPs) have attracted wide attention due to their surface effects, quantum size effects, quantum tunneling effects, and dielectric confinement effects. Guo et al. reported (Science China Chemistry, 2011, 54: 1640-1644) that metal oxide nanoparticles (MONPs) such as zinc oxide (ZnO), manganese dioxide (MnO2), indium oxide (In2O3), and titanium dioxide (TiO2) have an enhanced effect on the ECL of luminol, and the enhanced ECL signal is attributed to the adsorption of luminol on the surface of MONPs and the generation of ROS.

[0005] CN 109490284A discloses a dual catalytic luminol electrochemiluminescent biosensor based on gold nanoparticles and titanium dicarbide MXenes, which combines a multi-site recognition strategy and the reducibility of a new two-dimensional material-Ti3C2MXenes to form AuNPs-MXenes as an ECL signal probe, and designs a high-sensitivity electrochemiluminescent sensing platform to detect Hela exosomes. In this strategy, SA polymer molecules can provide a multi-site recognition interface to capture more aptamers to improve the capture efficiency of exosomes, which helps to improve the sensitivity of the biosensor and AuNP-MXenes can dually catalyze luminol ECL signal enhancement. Based on these two dual amplification strategies, a highly sensitive and highly selective biosensor is obtained to detect exosomes.

[0006] Nanozymes are an emerging multifunctional nanomaterial that can simulate the various catalytic properties of natural enzymes. However, dispersed free nanozymes will lead to a decrease in the local concentration of nanozymes and their intermediates, thereby limiting their catalytic performance. The stability and catalytic activity of the enzyme can be improved by fixing the nanozymes on a carrier or confining the nanozymes in a closed environment to construct a confined nanocatalytic reactor. For example, researchers confined MOF-818 nanozymes to the pores of macroporous tungsten trioxide (p-WO3), and the catalytic activity of the in situ assembled MOF-818 on 3,5-di-tert-butylcatechol (3,5-DTBC) can be adjusted by changing the pore size of p-WO3. Only in the p-WO3 pores with appropriate pore size can MOF-818 show high affinity and good catalytic activity for 3,5-DTBC.

[0007] At present, there are no reports on the use of metal oxide nanozymes as co-reactant promoters to enhance the electrochemiluminescence of luminol. Summary of the invention

[0008] In order to solve the problems of low detection sensitivity and great detection difficulty of tumor markers in serum samples, the present invention adopts nanochannel confined cobalt tetroxide (Co3O4) nanozyme as a co-reactant promoter to sensitize the ECL signal of luminol.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] A nanozyme-sensitized electrochemiluminescent immunobiosensor, comprising a three-electrode system of an immunoelectrode, a reference electrode, and a counter electrode, wherein the detection solution of the electrochemiluminescent immunosensor comprises luminol and hydrogen peroxide when used;

[0011] The immunoelectrode is an electrode of confined Co3O4 nanozyme, which is obtained by covalently fixing tumor marker antibodies and blocking non-specific sites.

[0012] The present invention improves the ECL signal of the luminol-H2O2 system based on the confined Co3O4 nanozyme in SNF. SNF has an ultra-small and uniform nanochannel array, which confines the Co3O4 nanozyme in the nanochannel, improves the stability of the nanozyme while enhancing its pseudo-peroxidase activity, thereby sensitizing the ECL signal of the luminol-H2O2 system. In addition, the outer surface of SNF is easy to modify, and after modification with epoxy groups, antibodies can be covalently fixed to prepare an immune recognition interface. When the detection object tumor marker is present in the solution, the antibody on the immune recognition interface can specifically capture the tumor marker to form an immune complex. The steric hindrance effect caused by the immune complex will reduce the diffusion of luminol and H2O2 to the electrode surface, thereby reducing the ECL signal. Based on the signal gating phenomenon caused by this immune recognition, ECL detection of tumor markers in human samples can be achieved.

[0013] The immunoelectrode preparation specifically comprises the following steps:

[0014] Step 1, immersing the SNF modified electrode in a CoSO4 solution, and electrodepositing to obtain a Co3O4 confined electrode Co3O4@SNF / ITO;

[0015] Step 2, immersing the confined Co3O4 electrode in a siloxane solution containing epoxy groups and allowing it to react to obtain an epoxy-functionalized confined electrode Co3O4@O-SNF / ITO;

[0016] Step 3, immersing the epoxy-functionalized confined electrode Co3O4@O-SNF / ITO in a buffer solution containing tumor marker antibodies for incubation, and then immersing it in a bovine serum albumin solution (BSA) for incubation after rinsing to obtain the immunoelectrode.

[0017] In the present invention, Co3O4 nanozymes are confined in the porous material of SNF, and the porous material is used to confine the nanomaterial, which can significantly improve the stability of the nanomaterial. In addition, the porous material provides a dispersion space, which can effectively increase the active sites of the nanomaterial, thereby improving its catalytic activity and stability.

[0018] The solvent used in the CoSO4-containing solution is water, the solvent used in the antibody solution is water, and the solvent used in the epoxy-containing siloxane solution is ethanol or isopropanol;

[0019] The SNF modified electrode is any one of an SNF modified indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode;

[0020] The preparation method of the SNF modified electrode comprises the following steps: A silicon dioxide nanoarray film is grown on the electrode by any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method.

[0021] Specifically, using The method for preparing SNF modified electrode by solution growth method comprises the following steps:

[0022] Sodium hexadecyl sulfonate (CTAB) was used as a template surfactant, dissolved in a water-ethanol solution, and ammonia and an organosiloxane precursor were added to obtain a precursor solution for growing SNF;

[0023] Inserting the electrode into the precursor solution for incubation, removing, washing and drying to obtain a modified electrode containing micelle (SM) template in the pores;

[0024] The micelles can be removed by immersing the electrode in a Cl-ethanol solution and stirring to obtain a SNF-modified electrode with open pores.

[0025] The method for preparing SNF modified electrode by EASA method comprises the following steps:

[0026] An organosiloxane precursor is added to a CTAB aqueous solution to obtain a precursor solution, an electrode is inserted into the precursor solution, a negative voltage is applied to the electrode to grow SNF, and the electrode is taken out, cleaned, and dried to obtain a modified electrode containing an SM template in the pores;

[0027] The micelles can be removed by immersing the electrode in a Cl-ethanol solution and stirring to obtain a SNF-modified electrode with open pores.

[0028] The molar concentration of CoSO4 in the CoSO4 solution in step 1 is 0.1-0.2M, the electrodeposition time is 1-7s, and the electrodeposition voltage is +1.0-2.0V; preferably, the deposition time is 3-6s, and more preferably, the deposition time is 5s.

[0029] In step 2, the molar concentration of GPTMS in the GPTMS solution is 2.0 mM to 3.0 mM, and the reaction is allowed to stand for 30-60 minutes at a temperature of 20-40°C.

[0030] In step 3, the concentration of the tumor marker antibody in the buffer solution containing the tumor marker antibody is 1 to 20 μg / mL, and the buffer solution is a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution; the solute concentration in the buffer solution is 0.005 to 0.5 mol / L, and the pH is 6.5 to 7.5.

[0031] The epoxy group siloxane includes any one or more of 3-aminopropyltriethoxysilane, (3-glycidylpropoxy)trimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane.

[0032] In step 3, the incubation temperature is 36-38° C. for 30-90 min.

[0033] Preferably, each step is rinsed with water or buffer after completion to remove excess undeposited or unincubated solution;

[0034] The molar concentration of luminol in the detection solution is 5-300 μmol / L, and the molar concentration of hydrogen peroxide is 10-100 μmol / L.

[0035] The present invention also provides a method for constructing the nanozyme-sensitized electrochemiluminescent immunoassay biosensor, comprising the steps of:

[0036] Step 1, immersing the SNF modified electrode in a CoSO4 solution, and electrodepositing to obtain a Co3O4 confined electrode Co3O4@SNF / ITO;

[0037] Step 2, immersing the confined Co3O4 electrode into a siloxane solution containing epoxy groups and allowing it to react to obtain an epoxy-functionalized confined electrode Co3O4@O-SNF / ITO; the organosiloxane containing epoxy groups can react with the silanol groups on the SNF and covalently bond to the SNF, and the epoxy groups can be used for the subsequent covalent immobilization of antibodies.

[0038] Step 3, immersing the epoxy-functionalized confined electrode Co3O4@O-SNF / ITO in a buffer solution containing tumor marker antibodies for incubation, and then immersing it in a bovine serum albumin solution (BSA) for incubation after rinsing to obtain the immunoelectrode;

[0039] Step 4: Using the three-electrode system of the immunoelectrode, the reference electrode and the counter electrode, and the electrolyte solution containing luminol and hydrogen peroxide as the detection solution, the electrochemiluminescent immunobiosensor is constructed.

[0040] The present invention also provides the use of the nanozyme-sensitized electrochemiluminescent immunobiosensor in the detection of tumor markers for non-diagnostic purposes, comprising the steps of:

[0041] The immunoelectrode is incubated with the sample solution containing tumor markers and then rinsed to obtain the immune complex modified electrode; the incubation temperature is 36-38°C for 30-90 minutes. The immune complex modified electrode is used as the modified electrode, and a three-electrode system is formed with a reference electrode and a counter electrode. The electrochemical luminescence signal is detected using an electrolyte solution containing luminol and hydrogen peroxide as the detection solution.

[0042] The detection principle is as follows: when tumor markers are present in serum samples, tumor marker antibodies bind to tumor markers, and the large immune complexes bound to the electrode interface will produce a significant steric hindrance effect on the diffusion and mass transfer of luminol and hydrogen peroxide, thereby changing the ECL signal of the electrode. Therefore, according to the quenching degree of the electrochemiluminescence signal, electrochemiluminescence detection of tumor markers can be achieved.

[0043] The sample to be tested contains glucose, non-target tumor markers, and one or more interfering compounds in inorganic salts.

[0044] Preferably, the sample solution to be tested includes a standard solution or a serum sample containing a tumor marker.

[0045] The detection limit of the electrochemical detection method is 18.5 fg mL -1 the following.

[0046] The triggering luminescence potential of the electrochemiluminescence immunosensor is 0.4-0.6V.

[0047] This detection method is used to detect tumor markers for non-diagnostic purposes, and can be used to study the relevant mechanisms of anti-tumor immune response, detect residual tumor markers after drug action, etc.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The SNF-based confined Co3O4 nanozyme of the present invention improves the ECL signal of the luminol-H2O2 system. SNF has an ultra-small and uniform nanochannel array, which confines the Co3O4 nanozyme in the nanochannel, improves the stability of the nanozyme and enhances its pseudo-peroxidase activity, thereby sensitizing the ECL signal of the luminol-H2O2 system.

[0050] (2) The outer surface of the SNF of the present invention is easy to modify. After modification with epoxy groups, antibodies can be covalently fixed to prepare an immune recognition interface. When the test object AFP is present in the solution, the antibodies on the immune recognition interface can specifically capture AFP to form an immune complex. The steric hindrance effect caused by the immune complex will reduce the diffusion of luminol and H2O2 to the electrode surface, thereby reducing the ECL signal. Based on the signal gating phenomenon caused by this immune recognition, ECL detection of AFP in human serum can be achieved. When the immunosensor of the present invention is used for detection, it only needs to incubate the immunoelectrode with the sample to be tested to measure the electrochemical signal, without the need for a sandwich immunoassay mode.

[0051] (3) The present invention is a study on the use of metal oxide nanozymes as co-reactant promoters to enhance the electrochemiluminescence of luminol. Co3O4 nanozymes are generated in situ and can significantly increase the ECL signal of the luminol and H2O2 system. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 (A) is a top view transmission electron microscope (TEM) photo of the ITO electrode (SNF / ITO) with silicon dioxide nanopores in Example 1. (B) is a scanning electron microscope (SEM) photo of the cross section of the SNF-modified ITO electrode. (C) is a top view SEM of SNF / ITO.

[0053] Figure 2 CV graphs of ITO, SM@SNF / ITO, and SNF / ITO in Example 1 in 50 mM potassium hydrogen phthalate (KHP) solution, 0.5 mM K3[Fe(CN)6](A), [Ru(NH3)6]Cl3(B), and FcMeOH(C) solutions.

[0054] Figure 3 SEM images of ITO (A), SNF / ITO (B) and Co3O4@SNF / ITO (C) in Example 1.

[0055] Figure 4 The scanning rate of Co3O4@SNF / ITO in 1M NaOH aqueous solution is 50mV s -1 CV curves at 40 ℃. (B) XPS spectra of SNF / ITO and Co3O4@SNF / ITO. (C) High-resolution Co 2p spectrum.

[0056] Figure 5 (A) SEM image of Co3O4@ITO in Example 1. (B) SEM image of Co3O4@SNF / ITO, where SNF is dissolved and removed by NaOH solution.

[0057] Figure 6 (A) ECL signal diagram of ITO, SNF / ITO, Co3O4@ITO and Co3O4@SNF / ITO in 100μM luminol and 100μM H2O2 in Example 1. (B) ECL signal diagram of Co3O4@SNF / ITO in 100μM luminol and 100μM H2O2 over time. PMT = 600V. Scan rate 100mV s -1 .

[0058] Figure 7 (A) CV graph of Co3O4@SNF / ITO electrode in different solutions in Example 1, and the corresponding inset is the CV graph of SNF / ITO in different solutions, with a scan rate of 50 mV / s. (B) CV graph of SNF / ITO and Co3O4@SNF / ITO in PBS (0.01 M, pH = 7.4), with a scan rate of 50 mV-1s.

[0060] Figure 8 UV-visible absorption spectra of different systems in (A) of Example 1. All measurements were performed in 0.1 M NaAc / HAc buffer solution (pH 4.0) containing 1 mM H2O2 and 0.25 mM TMB. (B) Different ROS capture agents (BQ: 100 μM; TBA: 100 μg mL -1 ), absorbance value of Co3O4@SNF / ITO-TMB-H2O2 system (λ=652nm). (C) ECL signal diagram of SNF / ITO and Co3O4@SNF / ITO in the presence of different ROS scavengers. (D) ECL mechanism based on luminol ternary system.

[0061] Fig. 9 In the presence of 100 μM luminol and 100 μM H2O2, the following results were obtained for SNF / ITO, Co3O4@SNF / ITO, Co3O4@O-SNF / ITO, and BSA / Ab / Co3O4@O-SNF / ITO (20 μg mL -1 ),AFP / BSA / Ab / Co3O4@O-SNF / ITO(1pg mL -1 ) and AFP / BSA / Ab / Co3O4@O-SNF / ITO (100 pg mL -1 ) ECL signal diagram. PMT = 600 V. Scan rate 100 mV s -1 Different electrodes in the presence of 2.5mM Fe(CN)6 3- / 4- CV (B) and EIS (C) graphs in 0.1 M KCl solution. The scan rate was 50 mV s-1 .

[0062] Fig.10 The Co3O4 deposition time (A), antibody concentration (B), and antibody (C) and antigen (D) incubation times in Example 1 were optimized.

[0063] Fig.11 (A) ECL signal of BSA / Ab / Co3O4@O-SNF / ITO electrode incubated with different concentrations of AFP in the presence of 100 μM luminol and 100 μM H2O2 in Example 1. PMT=600 V. Scan rate: 100 mV / s. (B) Linear calibration curve for detecting AFP.

[0064] Fig.12 The anti-interference ability of the ECL immunosensor (A) in Example 1 to different species. + 、Na + The concentration was 5 mM, the concentrations of glucose, UA, and GSH were 1 mM, and the concentrations of CEA, S100, and PSA were 10 ng mL -1 , CA19-9 concentration is 1mU mL -1 , AFP concentration was 1 ng mL -1 (B) Six BSA / Ab / Co3O4@O-SNF / ITO electrodes were prepared in parallel to detect 100 pg mL -1 ECL signal diagram of AFP.

[0065] Fig.13 (A) Comparison of the two methods for determining AFP content in Example 1. (B) The linear relationship between the quantitative results of AFP in plasma samples by this method and Roche electrochemiluminescence instrument. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0067] The raw materials used in the following specific embodiments are all purchased from the market.

[0068] Example 1

[0069] The process of using a silica nanoporous membrane modified electrode with nanochannel-confined Co3O4 nanozyme to covalently immobilize the alpha-fetoprotein (AFP) antibody on the outer surface to prepare an immunoelectrode and detecting AFP in serum samples by electrochemiluminescence:

[0070] (1) SNF / ITO Solution growth method preparation

[0071] First, 0.1600 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 70 mL of water and 30 mL of ethanol solution, and stirred for 5 min until the CTAB was completely dissolved; then, 100 μL of 10% ammonia water and 80 μL of tetraethoxysilane (TEOS) as a silicon source were added under stirring, and stirring was continued for 5 min until the solution became clear and transparent without bubbles, and the solution was carefully transferred to a staining jar.

[0072] Immerse clean ITO glass (2.5×5cm) in the precursor solution, seal the staining jar, and place it in a 60℃ water bath for 24h. After the growth is completed, take out the ITO electrode, rinse it with a large amount of deionized water, blow dry it with nitrogen, and age it overnight at 100℃ in an oven to obtain a modified electrode containing micelle (SM) templates in the pores, named SM@SNF / ITO. The prepared electrode is cut and its electrode area is controlled to 0.5×1cm with insulating glue as a working electrode. The electrode is immersed in 0.1M HCl-ethanol solution and stirred for 5min to remove the micelles, and the SNF / ITO electrode with open pores is obtained.

[0073] (2) Preparation of Co3O4@SNF / ITO

[0074] Co3O4@SNF / ITO was prepared by confining Co3O4 in SNF by one-step electrodeposition. The specific method is as follows:

[0075] The SNF / ITO was immersed in a CoSO4 (0.2 M) aqueous solution and a potential of +1.5 V was applied for 5 s to prepare the Co3O4@SNF / ITO electrode.

[0076] (3) Preparation of immunosensor

[0077] Co3O4@SNF / ITO was used as the substrate electrode, and γ-glycidyloxypropyltrimethylsilane (GPTMS) was used as a crosslinker to covalently crosslink the antibody and SNF layers. GPTMS was incubated before removing the micelles to avoid a large amount of GPTMS modification on the inner wall of the pores, which would hinder mass transfer.

[0078] The specific experimental steps are as follows: immerse the Co3O4@SNF / ITO electrode in an ethanol solution of GPTMS (2.26 mM), react at room temperature for 1 hour, modify the SNF surface with GPTMS through silanization reaction, and wash the electrode with deionized water to obtain a Co3O4@O-SNF / ITO electrode.

[0079] Secondly, AFP antibody (20 μg mL -1 , 40μL) was drop-coated on the surface of the Co3O4@O-SNF / ITO electrode, incubated at 37°C for 90min, washed thoroughly with PBS (0.01M, pH=7.4) to remove unbound antibodies, and blown dry to obtain the Ab / Co3O4@O-SNF / ITO electrode. Finally, the electrode prepared in the previous step was incubated with bovine serum albumin (BSA) solution (1%, 40μL) at room temperature for 15min to block nonspecific sites. Finally, it was washed thoroughly with PBS (0.01M, pH=7.4), blown dry, and the immunosensor was obtained, named BSA / Ab / Co3O4@O-SNF / ITO electrode.

[0080] (4) AFP electrochemiluminescence detection

[0081] In order to detect AFP, the BSA / Ab / Co3O4@O-SNF / ITO electrode was incubated with 50 μL of AFP of different concentrations at 37 °C for 90 min, and the electrode was slowly rinsed with PBS (0.01 M, pH = 7.4) solution to wash away the unbound AFP. The obtained electrode was recorded as AFP / BSA / Ab / Co3O4@O-SNF / ITO.

[0082] The obtained electrode was subjected to ECL detection, and the electrolyte solution used for the test was a PBS (0.01M, pH=7.4) solution containing 100 μM luminol and 100 μM H2O2. The ECL process was triggered by continuous CV scanning, and the CV scanning potential range was 0-0.8V, and the scanning rate was 0.1V / s.

[0083] In step 1, Electron microscopy of the ITO electrode modified with silica nanopores (SNF / ITO) prepared by the solution growth method Figure 1 shown.

[0084] Figure 1 (A) TEM top view of the ITO electrode modified with silica nanopores (SNF / ITO). The film shows a complete flat film structure without cracks. The pores represented by the bright spots are arranged in a random worm-like manner. The diameter of the pores is between 2-3 nm, and the porosity is about 75,000 pores / μm. 2 . Figure 1(B) shows the TEM image of the SNF / ITO cross section, which shows the long-range order of SNF channels and a thickness of about 86 nm. Figure 1 (C) shows a scanning electron microscope (TEM) image of the cross section of SNF / ITO. It can be seen that the electrodes are SNF layer, ITO layer and glass layer from top to bottom, and the thickness of SNF is 86nm.

[0085] Figure 2 The CV graphs of the electrodes ITO, SM@SNF / ITO, and SNF / ITO in the above steps in 50 mM potassium hydrogen phthalate (KHP) solution, 0.5 mM K3[Fe(CN)6](A), [Ru(NH3)6]Cl3(B), and FcMeOH(C) solutions. Figure 2 As shown in (AC), the SM@SNF / ITO electrode containing the surfactant template has no obvious Faraday current in the K3Fe(CN)6 and Ru(NH3)6Cl3 probes, indicating that the SM@SNF / ITO is intact without cracks or defects. On the contrary, the hydrophobic neutral probe FcMeOH can reach the electrode surface through micelle enrichment to generate redox signals. This process consumes a certain amount of energy, so when the redox reaction occurs, its peak potential shifts toward the positive potential.

[0086] For the SNF / ITO electrode with the template molecule removed, the negatively charged K3Fe(CN)6 showed an electrochemical signal lower than that of the bare ITO electrode, indicating that the probe was repelled, while the positively charged Ru(NH3)6Cl3 probe showed a peak current higher than that of the bare electrode. This demonstrates the charge-selective permeability of the SNF nanochannel. This obvious charge-selective permeability is attributed to the presence of numerous silanol groups with pKa~2 on the surface of SNF, which are ionized in a neutral solution environment, making the pores negatively charged, and can strongly repel anions and attract cations through electrostatic interactions. In addition, the reduction peak current of SNF / ITO in FcMeOH is greater than the oxidation peak current, because the oxidation products of FcMeOH molecules are positively charged and can be enriched in the pores, thus showing a higher reduction signal.

[0087] Figure 3 SEM images of ITO (A), SNF / ITO (B) and Co3O4@SNF / ITO (C). Figure 3 As shown in (AB), the ITO electrode and the SNF / ITO electrode without Co3O4 deposition have a flat surface. Figure 3 As shown in (C), there is no obvious change in the electrode surface morphology after the deposition of Co3O4, indicating that no large particles of nanomaterials are formed on the outer surface of SNF / ITO.

[0088] Figure 4The scanning rate of Co3O4@SNF / ITO in 1M NaOH aqueous solution is 50mV s -1 CV curves at 40 ℃. (B) XPS spectra of SNF / ITO and Co3O4@SNF / ITO. (C) High-resolution Co 2p spectrum.

[0089] like Figure 4 As shown in (A), two pairs of reversible redox peaks can be observed. This is due to the electrochemical oxidation and reduction reactions of cobalt compounds. Co3O4 is first oxidized to CoOOH in the potential range of 0.1-0.2V, and then oxidized to CoO2 in the range of 0.4-0.6V, and CoO2 can be reversibly reduced to CoOOH and Co3O4. XPS was used to characterize the elemental composition of the Co3O4@SNF / ITO electrode. Figure 4 (B) shows the XPS spectra of SNF / ITO and Co3O4@SNF / ITO. It can be observed that compared with SNF / ITO, the Co3O4@SNF / ITO electrode shows Co 3p and Co 2p features at around 64eV and 779eV. Figure 4 (C) is the high-resolution Co 2p spectrum. 779.4 eV is the characteristic peak of Co3O4 material. More importantly, Co 2p 1 / 2 and Co 2p 2 / 3 The energy band gap of 14.6 eV between them is also a characteristic mark in the XPS spectrum of Co3O4.

[0090] Figure 5 (A) is the SEM image of Co3O4@ITO in Example 1. (B) is the SEM image of Co3O4@SNF / ITO, where (A) Co3O4@ITO is obtained by immersing Co3O4@SNF / ITO in 0.5M NaOH for 5 minutes, and SNF is dissolved and removed by the NaOH solution.

[0091] like Figure 5 As shown in (A), the SEM image of Co3O4 directly electrodeposited on the ITO electrode also shows that a large area of ​​nanoparticles of different sizes appear on the electrode surface. Depending on the degree of agglomeration, the average size of the particles is about 100-200nm. Element mapping confirms that the material structure is Co3O4 nanoparticles. Figure 5As shown in (B), a large number of nanoparticles appeared on the surface of the ITO electrode, with an average size of about 50-100nm. Element mapping confirmed that the substance was Co3O4. It is speculated that the surface energy of the nanoparticles is high, and they are very easy to agglomerate in the absence of a protective agent. The two-dimensional rigid structure of the nanochannel array can limit the growth of nanoparticles and can stably confine them in the nanochannel. When the silicon dioxide film on the surface is dissolved, Co3O4 agglomerates without the protection of the nanopores. The above results prove that the electrodeposited Co3O4 is successfully confined inside the nanopores.

[0092] Figure 6 (A) ECL signal diagram of ITO, SNF / ITO, Co3O4@ITO and Co3O4@SNF / ITO in 100μM luminol and 100μM H2O2 in Example 1. (B) ECL signal diagram of Co3O4@SNF / ITO in 100μM luminol and 100μM H2O2 over time. PMT = 600V. Scan rate 100mV s -1 .

[0093] like Figure 6 As shown in (A), the ITO electrode has an electrochemical luminescence signal of 6200. When modified with a layer of mesoporous silica nanochannel film, its ECL signal drops to 2784. This is because in the buffer system of pH = 7.4, the SNF surface contains many silanols with pKa ~ 2, which are ionized and the pores are negatively charged, which is conducive to the luminescent probe luminol － There is a repulsive effect, which prevents the probe from reaching the electrode surface for further reaction. After depositing Co3O4, the ECL signals of Co3O4@ITO and Co3O4@SNF / ITO increased to 10733 and 17009, respectively, an increase of 1.7 times and 6.1 times, proving that the presence of Co3O4 can effectively promote the ECL signal intensity of the luminol-H2O2 system, but the degree of increase is different. It is speculated that when nanochannels exist, the growth of nanoparticles can be well restricted to obtain Co3O4 with smaller size and larger specific surface area. Co3O4 deposited directly on the bare ITO electrode is very easy to agglomerate due to the influence of surface energy, which corresponds to the previous electron microscope image. Figure 6 As shown in (B), the prepared Co3O4@SNF / ITO has a stable and high ECL signal, which indicates that the prepared Co3O4@SNF / ITO is a good platform for the subsequent construction of immunosensors.

[0094] In a typical anode luminol-H2O2 system, H2O2 acts as a co-reactant to produce various reactive oxygen species. The generated ROS react with the luminol anion radicals generated by oxidation on the electrode surface to generate excited substances, which release energy and emit blue light. Therefore, improving the oxidation efficiency of the luminescent probe on the electrode and the efficiency of the co-reactant in generating ROS are the key to improving the luminol-H2O2 anode luminescence. The above experiments prove that Co3O4 has a significant sensitization effect on the ECL signal of the luminol–H2O2 system, and further explore its sensitization mechanism.

[0095] The Co3O4@SNF / ITO electrode was placed in different solutions for electrochemical scanning, including PBS buffer, 0.01M PBS buffer (pH=7.4) with 100μM luminol and 100μM H2O2, and the scanning rate was 50mV / s. Figure 7 As shown, (A) CV diagram of Co3O4@SNF / ITO electrode in different solutions, the corresponding inset is the CV diagram of SNF / ITO in different solutions, (B) CV diagram of SNF / ITO and Co3O4@SNF / ITO in PBS (0.01M, pH=7.4), with a scanning rate of 50mV-1s.

[0097] like Figure 7 As shown in (A), the SNF / ITO electrode cannot respond to the Raday current in PBS. After confining Co3O4, the Co3O4@SNF / ITO electrode has a current response of 9.9μA at 0.8V. This is because Co3O4 promotes the oxygen evolution of water. On this basis, 100μM luminol was added, and the current signal increased to 20μA, minus the 3.5μA current response of the substrate electrode SNF / ITO to luminol. We believe that the remaining 6.6μA is caused by the electrocatalytic oxidation of luminol by Co3O4. Similarly, we further explored whether Co3O4 has the same electrocatalytic effect on H2O2. The SNF / ITO electrode had no current response in both PBS and 100μM H2O2, while the Co3O4@SNF / ITO electrode had a current response of 17μA at 0.8V in a 100μM H2O2 solution. After deducting the 9.9μA contribution of the Co3O4@SNF / ITO electrode itself to water oxygen evolution, the remaining 7.1μA was caused by the electrocatalytic oxidation of H2O2 by Co3O4.

[0098] like Figure 7As shown in (B), by comparing the charging current at 0.2 V, it is found that the electrode with confined Co3O4 has an increased electroactive area of ​​26.9% compared with the SNF / ITO electrode. Therefore, the electrocatalytic oxidation of luminol and H2O2 by Co3O4 and the increase in the electroactive area of ​​the electrode are the main reasons for the enhanced ECL signal of the luminol-H2O2 system.

[0099] like Figure 8 As shown in (A), TMB was used as the chromogenic substrate to study whether Co3O4 has pseudoperoxidase activity in the presence of H2O2. After the three electrodes reacted in situ in a mixture of TMB and H2O2 for a certain period of time, the absorbance at a wavelength of 652 nm was monitored. It can be observed that the changes in color and absorbance of the solutions after the reactions of TMB+H2O2 (i) and SNF / ITO+TMB+H2O2 (ii) are negligible, while the color of the solution after the reaction of Co3O4@SNF / ITO+TMB+H2O2 (iii) turns blue significantly and the absorbance of the oxidation product oxTMB at 652 nm is also significantly increased, indicating that Co3O4 has good pseudoperoxidase activity.

[0100] Next, different free radical scavengers were used to investigate the types of ROS produced in this process. 0.5 mM of p-benzoquinone (BQ) or tert-butyl alcohol (TBA) was added to the luminol hydrogen peroxide solution. Figure 8 As shown in (B), when O2 is added ·- After the capture agent benzoquinone (BQ) was added, the absorbance decreased significantly. After the addition of ·OH capture agent tert-butyl alcohol (TBA), the absorbance of the system remained basically unchanged. This indicates that the ROS species in the enzymatic process is O2 ·- .

[0101] like Figure 8 (C) is a comparison of the ECL signals of different electrodes after the ECL process was triggered by continuous cyclic voltammetry (CV) in an environment containing luminol and H2O2 and after the addition of quencher. It can be seen from the figure that the ECL of SNF / ITO and Co3O4@SNF / ITO electrodes was 100% quenched after the addition of p-benzoquinone, and the type of ROS generated was still O2 ·- , indicating that the type of ROS produced by the electrocatalytic oxidation of H2O2 by Co3O4 is O2 ·- Therefore, the above experiments prove that Co3O4 can be used as an electron enhancement material to electrocatalyze the oxidation of H2O2 and luminol, and can also be used as a pseudo-peroxidase to catalyze the production of O2 from H2O2. ·- .

[0102] The reaction formula of the ECL reaction mechanism of the ternary system of luminol+H2O2+Co3O4 is as follows:

[0103] LH2-H + →LH -

[0104]

[0105] AP 2-* →AP 2- +hv

[0106] First, luminol loses protons in a weakly alkaline solution to become luminol - (LH - ), LH - When it reaches the electrode surface, it loses electrons and protons under the catalysis of Co3O4 to generate luminol ·- (L ·- ), and H2O2 produces a large amount of O2 under the dual action of Co3O4 enzyme catalysis and electrocatalysis. ·- , L ·- With O2 ·- The excited state AP 2-* , returns to the ground state accompanied by luminescence.

[0107] In the presence of 100 μM luminol and 100 μM H2O2, the prepared electrodes SNF / ITO, Co3O4@SNF / ITO, Co3O4@O-SNF / ITO, BSA / Ab / Co3O4@O-SNF / ITO (20 μg mL -1 ),AFP / BSA / Ab / Co3O4@O-SNF / ITO(1pg mL -1 ) and AFP / BSA / Ab / Co3O4@O-SNF / ITO (100 pg mL -1 ) Test ECL signal diagram. PMT = 600V. Scan rate is 100mV s -1 Different electrodes in the presence of 2.5mM Fe(CN)6 3- / 4- CV (B) and EIS (C) graphs in 0.1 M KCl solution. The scan rate was 50 mV s -1 .

[0108] like Fig. 9 As shown in (A), the ECL signal of SNF / ITO electrode in the luminol-H2O2 system is the lowest. After Co3O4 is deposited in the nanochannel, the ECL signal of Co3O4@SNF / ITO is significantly enhanced. This is attributed to the catalysis of Co3O4 on luminol and H2O2 to produce a large amount of luminol ·- and O2 ·-, which enhanced the electrochemiluminescence of luminol. With the modification of epoxy groups, the ECL signal of Co3O4@O-SNF / ITO was slightly lower than that of Co3O4@SNF / ITO. This is because the modification of epoxy functional groups will affect the charge and surface state of SNF. After further modification of Ab and blocking of nonspecific sites, the non-conductive protein layer formed on the electrode surface will hinder the mass transfer and electron transfer of the probe, so the ECL signal of BSA / Ab / Co3O4@O-SNF / ITO is significantly reduced. When AFP binds to Ab, the immune complex formed further hinders the diffusion of luminol and H2O2. With the increase of AFP concentration, the ECL signal of the immunosensor gradually decreases. The above experimental results prove the feasibility of the constructed immunosensor.

[0109] like Fig. 9 As shown in (B), CV and EIS were used to characterize the modified electrodes. 3- / 4- As a redox probe, the corresponding CV curves under different modified electrodes were recorded. With the gradual modification of the electrode, the steric hindrance formed on the SNF surface gradually increased, the conductivity decreased, and the redox reaction activity of the probe gradually decreased. Fig. 9 As shown in (C), similar phenomena can be observed in the EIS data. The EIS spectrum consists of a high-frequency semicircular part and a low-frequency linear part. The illustration on the right side of the figure is an equivalent circuit schematic, which includes solution resistance (Rs), double-layer capacitance (Cdl), Warburg impedance (Zw) and apparent charge transfer resistance (Rct); where Rct is equal to the equivalent diameter of the semicircular part in the high-frequency region. Among them, Co3O4@SNF / ITO has the lowest Rct (1625Ω). After modifying the epoxy group, Rct increases to 1693Ω. After further modifying the antibody and blocking the specific site, Rct increases to 2417Ω. Finally, after the target specifically binds to the antibody, Rct increases to 7081Ω, proving the feasibility of the constructed immunosensor.

[0110] The Co3O4 deposition time in step 2, the antibody concentration in step 3, the antibody concentration in step 4, and the antigen concentration in step 4 were changed separately to observe their effects on the ECL signal of the Co3O4@SNF / ITO electrode. Fig.10 As shown, Co3O4 deposition time (A), antibody concentration (B), antibody incubation time (C), and antigen incubation time (D).

[0111] like Fig.10As shown in (A), the ECL intensity of the Co3O4@SNF / ITO electrode obtained by electrodeposition at different times was compared. The results show that with the increase of Co3O4 deposition time, the ECL intensity of the electrode shows a trend of increasing first and then decreasing. Because as the Co3O4 deposition time increases, the deposition amount of Co3O4 will also increase, but too long a deposition time will lead to a large particle size of Co3O4 nanoparticles, resulting in pore blockage or deposition out of the pores, which will reduce the diffusion of the ECL luminophore and co-reactant in the nanochannel. Therefore, in order to achieve the highest ECL signal, the electrode obtained by depositing Co3O4 for 5 seconds was selected for subsequent experiments.

[0112] like Fig.10 As shown in (BD), the antibody dosage, antibody incubation time and AFP binding time were optimized, taking into account time and cost factors. The final selected antibody dosage was 20 μg / mL, the antibody incubation time was 90 min, and the AFP binding time was 90 min.

[0113] like Fig.11 (A) shows the ECL signal measured after the immunosensor was incubated with different concentrations of AFP. It can be seen that with the increase of AFP concentration, the number of immune complexes formed on the electrode surface increases, the electron transfer resistance gradually increases, the hindering effect on the diffusion of ECL luminophore and co-reactant increases, and the ECL signal of the electrode gradually decreases, thereby achieving quantitative detection of AFP. Fig.11 (B) is the corresponding linear fitting curve. The results show that in the range of 100fg / mL to 10ng / mL, the ECL signal (I ECL ) and the logarithm of AFP concentration (logC AFP ) shows a good linear relationship. The regression equation is ECL =–1345logC AFP (ng / mL)+4733(R 2 =0.997), and the LOD was 18.5 fg / mL. As a control, the performance of the immunosensor directly constructed using SNF / ITO electrodes to detect AFP was investigated. Fig.11 As shown in (B), the linear range of AFP detection is from 10 pg mL -1 to 10 ng mL -1 (I ECL =-246logC AFP +1402, R 2 =0.991). It can be seen that the introduction of Co3O4 as a nanocatalyst to enhance the ECL signal can significantly improve the detection linear range and sensitivity of the immunosensor.

[0114] Some common substances in serum and some common tumor markers including carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), glutathione (GSH), calcium binding protein β (S100) and inorganic ions, glucose (Glu) were selected as possible interferents to investigate the selectivity and anti-interference ability of the immunosensor. + 、Na + The concentration was 5 mM, the concentrations of glucose, UA, and GSH were 1 mM, and the concentrations of CEA, S100, and PSA were 10 ng mL -1 , CA19-9 concentration is 1mU mL -1 , AFP concentration was 1 ng mL -1 After these interfering substances were incubated in step 4, the ECL signal was measured. Fig.12 As shown in (A), none of the above interferents will cause significant changes in the ECL signal of the electrode. Only when AFP is present will the electrode show obvious signal changes. The above experiments prove that the prepared immunosensor has good selectivity and anti-interference ability.

[0115] The reproducibility of the immunosensor was studied, e.g. Fig.12 As shown in (B), the relative standard deviation of the ECL signal intensity of 6 electrodes prepared in the same batch after incubation with 100 pg / mL AFP was 1.3%, indicating that the sensor has good reproducibility.

[0116] like Fig.13 As shown in (AB), the constructed immunosensor was used to measure the AFP content in the actual serum samples of normal people and patients. The serum samples of 7 groups of patients and 7 groups of normal people were selected and diluted 50 times for detection. The test values ​​of this method were compared with the test values ​​of Roche electrochemiluminescence instrument. The comparison results showed that the two methods had good consistency.

Claims

1. A nanozyme-sensitized electrochemiluminescent immunoassay biosensor. It is characterized in that A three-electrode system comprising an immunoelectrode, a reference electrode and a counter electrode, wherein the detection solution of the electrochemiluminescent immunosensor comprises luminol and hydrogen peroxide when the electrochemiluminescent immunosensor is used; The immunoelectrode is confined Co 3 O 4 The nanozyme electrode is obtained by covalently fixing tumor marker antibodies and blocking nonspecific sites.

2. The nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to claim 1, It is characterized in that The immunoelectrode preparation specifically comprises the following steps: Step 1: Immerse the SNF-modified electrode in CoSO 4 In solution, confined Co is obtained by electrodeposition 3 O 4 Electrode Co 3 O 4 @SNF / ITO; Step 2: confine Co 3 O 4 The electrode was immersed in a siloxane solution containing epoxy groups and allowed to react to obtain an epoxy-functionalized confined electrode Co 3 O 4 @O-SNF / ITO; Step 3: Functionalize the epoxy-confined electrode Co 3 O 4 @O-SNF / ITO is immersed in a buffer solution containing tumor marker antibodies for incubation, and then immersed in a bovine serum albumin solution for incubation after being rinsed to obtain the immunoelectrode.

3. The nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to claim 2, It is characterized in that The CoSO 4 The solvent used in the solution is water, the solvent used in the antibody solution is water, and the solvent used in the epoxy group-containing siloxane solution is ethanol or isopropanol; The SNF modified electrode is any one of an SNF modified indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode; The preparation method of the SNF modified electrode comprises the following steps: A silicon dioxide nanoarray film is grown on the electrode by any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method.

4. The nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to claim 2, It is characterized in that Step 1 contains CoSO 4 CoSO in solution 4 The molar concentration is 0.1-0.2M, the electrodeposition time is 1-7s, and the electrodeposition voltage is +1.0-2.0V; In step 2, the molar concentration of GPTMS in the GPTMS solution is 2.0 mM to 3.0 mM, and the reaction is allowed to stand for 30-60 minutes at a temperature of 20-40° C.; The epoxy group siloxane includes any one or more of 3-aminopropyltriethoxysilane, (3-glycidylpropoxy)trimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane.

5. The nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to claim 2, It is characterized in that In step 3, the concentration of the tumor marker antibody in the buffer solution containing the tumor marker antibody is 1 to 20 μg / mL, and the buffer solution is a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution; the solute concentration in the buffer solution is 0.005 to 0.5 mol / L, and the pH is 6.5 to 7.

5.

6. The nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to claim 2, It is characterized in that In step 3, the incubation temperature is 36-38° C. for 30-90 min.

7. The nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to claim 1, It is characterized in that The molar concentration of luminol in the detection solution is 5-300 μmol / L, and the molar concentration of hydrogen peroxide is 10-100 μmol / L.

8. A method for constructing a nanozyme-sensitized electrochemiluminescent immunoassay biosensor according to any one of claims 1 to 7, It is characterized in that Includes steps: Step 1: Immerse the SNF-modified electrode in CoSO 4 In solution, confined Co is obtained by electrodeposition 3 O 4 Electrode Co 3 O 4 @SNF / ITO; Step 2: confine Co 3 O 4 The electrode was immersed in a siloxane solution containing epoxy groups and allowed to react to obtain an epoxy-functionalized confined electrode Co 3 O 4 @O-SNF / ITO; Step 3: Functionalize the epoxy-confined electrode Co 3 O 4 @O-SNF / ITO is immersed in a buffer solution containing tumor marker antibodies for incubation, and then immersed in a bovine serum albumin solution (BSA) for incubation after rinsing to obtain the immunoelectrode; Step 4: Using the three-electrode system of the immunoelectrode, the reference electrode and the counter electrode, and the electrolyte solution containing luminol and hydrogen peroxide as the detection solution, the electrochemiluminescent immunobiosensor is constructed.

9. Use of the nanozyme-sensitized electrochemiluminescent immunobiosensor according to any one of claims 1 to 7 in the detection of tumor markers for non-diagnostic purposes, It is characterized in that Includes steps: The immunoelectrode is incubated with a sample solution containing tumor markers and then rinsed to obtain an immune complex modified electrode; The immune complex modified electrode was used as the modified electrode, and a three-electrode system was formed with a reference electrode and a counter electrode. An electrolyte solution containing luminol and hydrogen peroxide was used as the detection solution for electrochemiluminescence signal detection.

10. Use of the nanozyme-sensitized electrochemiluminescent immunobiosensor according to claim 9 in the detection of tumor markers for non-diagnostic purposes, It is characterized in that The incubation temperature is 36-38° C., and the incubation time is 30-90 min. The sample to be tested contains one or more interfering compounds among glucose, non-target tumor markers, and inorganic salts.

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